The significance of the "leaky" tight junction might be understood better if cells of the epithelial monolayer possessed mechanisms to regulate molecular flow through the junction. To test this possibility, Necturus gallbladder, a representative leaky epithelium, was studied before, during, and after mucosal exposure to plant cytokinins and two other microfilament-active drugs, cytochalasin B and phalloidin. Concomitant with morphological changes in microfilaments, cytokinins induced rapid reversible increases in transepithelial resistance and potential difference (PD) and decreases in NaCl dilution potentials, with no change in the ratio of relative cell membrane resistances. Cytochalasin B (0.2-1.2 microM) and phalloidin (0.6-12.7 microM) caused similar changes in transepithelial resistance and PD. When the intramembranous structure of tight junctions was studied by freeze fracture, peak cytokinin-induced increments in transepithelial resistance were associated with more disorder in the strand meshwork resulting in a small increase in tight junction depth, but there was no evidence of de novo strand assembly. These studies suggest that permeability of the tight junction of Necturus gallbladder is subject to rapid reversible modulation, possibly under cytoskeletal control.
Chloride channels are present in the plasma and intracellular membranes of most cells. Previously, using the ligand indanyloxyacetic acid (IAA), we purified four major proteins from bovine kidney cortex membrane vesicles. These proteins gave rise to chloride channel activity when reconstituted into phospholipid vesicles. Two ofthese proteins (97 and 27 kDa) were found to be drug-binding proteins by N-terminal sequence analysis. Antibodies raised to the 64-kDa protein stained only this protein on immunoblots, and only this protein was present after purification on an immunoaffinity column. In addition, these same antibodies were able to deplete IAA-94 inhibitable chloride channel activity from solubilized kidney membranes. Of fractions obtained from the gel filtration ofsolubilized kidney membranes, only those containing this 64-kDa protein exhibited measurable chloride channel activity. Immunoblots of a variety of species and cell types, both epithelial and nonepithelial, revealed that this protein is ubiquitous and highly conserved. Immunocytochemistry in CFPAC-1 cells revealed staining for this protein on the apical plasma membrane and in the membranes of intracellular organelles. These results demonstrate that the integral membrane protein p64 is a component of chloride channels present in both epithelial plasma membrane and the membranes of intracellular organelles.
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